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Divergent response to LPS and bacteria in CD14-deficient murine macrophages
K J Moore1, L P Andersson, R R Ingalls
1Lipid Metabolism Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Abstract:
Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.
Insights
Mice lacking the CD14 protein show no inflammatory response to lipopolysaccharide (LPS). However, they still release inflammatory mediators when exposed to whole bacteria, indicating alternative immune pathways exist.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Gram-negative bacteria and lipopolysaccharide (LPS) trigger inflammatory responses linked to septic shock.
- The CD14 membrane protein initiates LPS-induced inflammatory signaling.
Purpose of the Study:
- To generate and characterize CD14-null mice to investigate the role of CD14 in inflammatory responses.
- To understand the compensatory mechanisms in the absence of CD14.
Main Methods:
- Generation of CD14-null mice by deleting the entire CD14 coding region.
- Assessing TNF-alpha and IL-6 production in macrophages via LPS and whole Escherichia coli stimulation.
- Utilizing FACS analysis for bacterial binding and uptake, and receptor blockade (neutrophil inhibitory factor) and phagocytosis inhibition (cytochalasin B).
Main Results:
- CD14-null macrophages failed to produce TNF-alpha and IL-6 in response to LPS, with impaired NF-kappaB and c-Jun N-terminal kinase pathway activation.
- Bacterial binding and uptake were unaffected in CD14-null macrophages.
- Whole E. coli stimulated TNF-alpha release in CD14-null macrophages, an effect partially mediated by CD11b/CD18 and dependent on phagocytosis.
Conclusions:
- CD14 is crucial for macrophage response to free LPS.
- Receptors like CD11b/CD18 can compensate for CD14's absence in response to whole bacteria.
- Phagocytosis plays a role in CD14-independent inflammatory responses to bacteria.